The present invention generally relates to compositions and methods for the detection of activities, such as enzymatic activities, using fluorescent compounds that are modified by the activity such that they exhibit a change in their sensitivity to quenching.
Fluorescent compounds have been used in the art to detect a wide variety of biological phenomenon, such as changes in ion concentration, specific binding reactions, subcellular localization, and enzymatic reactions. In the case of detecting changes in ion concentration, specific binding reactions, and subcellular localization, the fluorescent compound is used as a label to detect such specific binding or localization. In some cases, the fluorescence of the fluorescent compound is altered after an enzyme has acted on the fluorescent compound or a molecule binds with the fluorescent compound. For example, the activity of beta-galactosidase on the substrate fluorescein di-beta-D-galactopyranoside causes an increase in fluorescence of the substrate (Molecular Probes Catalogue, Sixth Edition, p.208 (1996)). Likewise, the action of beta-lactamase on CCF2-AM causes the compound to change fluorescence from green to blue due to an uncoupling of fluorescence resonance energy transfer (FRET) (Tsien and Zlokarnik, WO 96/30540, published Oct. 3, 1996). Protease activity can also be detected by the action of a protease on a fluorescent compound that uncouples FRET of the fluorescent compound (Tsien et al., WO 97/28261, published Aug. 7, 1997).
The detection of enzymatic reactions is important for the study of biological phenomenon, cellular biology, medical diagnostics, and drug discovery. Several classes of enzymes have been implicated in disease states, such as proteases for HIV and kinases for cancer. Drug discovery preferably uses living cells to detect compounds that can alter the activity enzymes involved in such disease states. However, ex vivo methods can also be used in drug discovery
Protein kinases and phosphatases have particularly been recognized as one of the more important general mechanism of cellular regulation. Protein phosphorylation commonly occurs on three major amino acids, tyrosine, serine or threonine. Changes in the phosphorylation state of these amino acids within proteins can regulate many aspects of cellular metabolism, regulation, grown and differentiation. Changes in the phosphorylation state of proteins, mediated through phosphorylation by kinases, or dephosphoryation by phosphatases, is a common mechanism through which cell surface signaling pathways transmit and integrate information into the nucleus. Given their key role in cellular regulation, it is not surprising that defects in protein kinases and phosphatases have been implicated in many disease states and conditions. For example, the over-expression of cellular tyrosine kinases such as the EGF or PDGF receptors, or the mutation of tyrosine kinases to produce constitutively active forms (oncogenes) occurs in many cancer cells (Durker et al. Nature Medicine 2:561-556 (1996)). Protein tyrosine kinases are also implicated in inflammatory signals, and defective Thr/Ser kinase genes have been demonstrated to be implicated in several diseases such as myotonic dystrophy, cancer and Alzheimer""s disease (Sanpei et al., Biochem. Biophys. Res. Commun. 212:341-346 (1995); Sperger et al., Neurosci. Lett. 197:149-153 (1995); Grammas et al., Neurobiology of Aging, 16:563-569 (1995); Govani et al., Ann. N.Y. Acad. Sci. 777:332-337 (1996)).
The involvement of proteases, protein kinases, protein phosphatases, and other classes of enzymes in disease states makes them attractive targets for the therapeutic intervention of drugs. In fact, many clinically useful drugs act on protein kinases or phosphatases. Examples include cyclosporin A, a potent immunosuppresent that binds to cyclophilin. This complex binds to the Ca++/calmodulin-dependent protein phosphatase type 2B (calcineurin), inhibiting its activity, and hence the activation of T cells. Inhibitors of protein kinase C are in clinical trials as therapeutic agents for the treatment of cancer (Clin. Cancer Res. 1:113-122 (1995)) as are inhibitors of cyclin dependent kinase (J. Mol. Med. 73:509-514 (1995)).
The number of known enzymes, such as kinases and phosphatases, are growing rapidly as the influence of genomic programs to identify the molecular basis for diseases have increased in size and scope. These studies are likely to implicate many more genes that encode enzymes that are involved in the development and propagation of diseases in the future, thereby making them attractive targets for drug discovery. However, current methods of measuring enzyme activity, such as protein phosphorylation and dephosphorylation, have many disadvantages which prevents or limits the ability to rapidly screen for drugs using miniaturized automated formats of many thousands of compounds. In the case of phosphatases and kinases, this is because current methods rely on the incorporation and measurement of 32P into the protein substrates of interest. In whole cells this necessitates the use of high levels of radioactivity to efficiently label the cellular ATP pool and to ensure that the target protein is efficiently labeled with radioactivity. After incubation with test drugs, the cells must be lysed and the protein of interest purified to determine its relative degree of phosphorylation. This method requires high numbers of cell, long preincubation times, careful manipulation, and washing steps to avoid artifactal phosphorylation or dephosphorylation. Furthermore, this approach requires purification of the target protein, and final radioactive incorporation into target proteins is usually very low, giving the assay poor sensitivity. Alternative assay methods, such as those based on phosphorylation-specific antibodies using ELISA-type approaches, involve the difficulty of producing antibodies that distinguish between phosphorylated and non-phosphorylated proteins, and the requirement for cell lysis, multiple incubations, and washing stages which are time consuming, complex to automate, and potentially susceptible to artifacts.
Fluorescent molecules are attractive as reporter molecules in many assay systems because of their high sensitivity and ease of quantification. Recently, fluorescent proteins have been the focus of much attention because they can be produced in vivo by biological systems and can be used to trace and monitor intracellular event without the need to be introduced into the cell through microinjection or permeabilization. The green fluorescent protein of Aequorea victoria is particularly interesting as a fluorescent indicator protein. A cDNA for the protein has been cloned (Prasher et al., Gene 111:229-233 (1992)). Not only can the primary amino acid sequence of the protein be expressed from the cDNA, but the expressed protein can fluoresce in cells in vivo.
Fluorescent proteins have been used as markers of gene expression, tracers of cell lineage, and as fusion tags to monitor protein localization within living cells (Rizzuto et al., Current Biol. 6:183-188 (1996)); Cubitt et al., TIBS 20:448-455 (1995); U.S. Pat. No. 5,625,048 to Tsien et al, issued Apr. 29, 1997). Furthermore, mutant versions of green fluorescent protein have been identified that exhibit altered fluorescence characteristics, including altered excitation and emission maxima, as well as excitation and emission spectra of different shapes. (Heim, Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994); Heim et al., Nature 373:663-665 (1995); U.S. Pat. No. 5,625,048, Tsien et al., issued Apr. 29, 1997; WO 97/28261 to Tsien et al, published Aug. 7, 1997; PCT/US 97/12400 to Tsien, filed Jul. 16, 1997; and PCT/US 97/14593 by Tsien, filed Aug. 15, 1997). These proteins add variety and utility to the arsenal of biologically based fluorescent indicators.
There is thus a need for assays for enzymes, such as those involved in protein phosphorylation, that are sensitive, simple to use, useful in living cells, and adaptable to high throughput screening methods. Preferably, such assays would not utilize radioactive materials so that the assays would be safe and not generate hazardous wastes. The present invention addresses these needs, and provides additional benefits as well.